(Chapter 8) Attachment site of the Gifsy-1 prophage in the S. enterica serovar Typhimurium chromosome.The four panels show the DNA sequences in the region of site-specific recombination between the phage (attP) and the chromosome (attB) and the left and right boundaries of the prophage (attL and attR). Black and white lettering indicates phage and chromosomal sequences, respectively. Purple boxes delimit a 14-bp sequence found duplicated at the prophage ends. Opposing arrows above this sequence indicate regions of dyad symmetry.

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(Chapter 8) Attachment site of the Gifsy-1 prophage in the S. enterica serovar Typhimurium chromosome.The four panels show the DNA sequences in the region of site-specific recombination between the phage (attP) and the chromosome (attB) and the left and right boundaries of the prophage (attL and attR). Black and white lettering indicates phage and chromosomal sequences, respectively. Purple boxes delimit a 14-bp sequence found duplicated at the prophage ends. Opposing arrows above this sequence indicate regions of dyad symmetry.

(Chapter 8) (A) Gene organization at the right end of the Gifsy-3 prophage (84) (GenBank accession no.AY633740). (B) Comparison of the 3′ end segment of the icd gene with and without integrated Gifsy-3 (GenBank accession no.AY633740).

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(Chapter 8) (A) Gene organization at the right end of the Gifsy-3 prophage (84) (GenBank accession no.AY633740). (B) Comparison of the 3′ end segment of the icd gene with and without integrated Gifsy-3 (GenBank accession no.AY633740).

(Chapter 8) Tandem array of prophage-related inserts in S. enterica. The diagram is based on data from references 11, 37, and 83 and from the S. enterica serovar Enteritidis strain LK5 genome sequence project at the University of Illinois, Urbana-Champaign.

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(Chapter 8) Tandem array of prophage-related inserts in S. enterica. The diagram is based on data from references 11, 37, and 83 and from the S. enterica serovar Enteritidis strain LK5 genome sequence project at the University of Illinois, Urbana-Champaign.

(Chapter 16) Schematic of novel putative extracellular protein encoded by the mefA element.The amino-terminal secretion signal sequence (SSS) is shown in red. Amino acids 21 to 713 correspond to a region that is rich in lysine, aspartic acid, and glutamic acid. Amino acids 714 to 1253 (shown in blue) are characterized by six identical 90-amino-acid blocks. Each of these six repeat regions has VTYPD and KEKEE amino acid sequence motifs, which are present in the GAS R28 protein and the P. falciparum MESA protein, respectively. An LP-KTG cell wall anchor motif (shown in yellow) is located at the carboxy terminus. Numbers in the protein schematic refer to amino acid residues. Reprinted from reference 7 with permission of the publisher.

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(Chapter 16) Schematic of novel putative extracellular protein encoded by the mefA element.The amino-terminal secretion signal sequence (SSS) is shown in red. Amino acids 21 to 713 correspond to a region that is rich in lysine, aspartic acid, and glutamic acid. Amino acids 714 to 1253 (shown in blue) are characterized by six identical 90-amino-acid blocks. Each of these six repeat regions has VTYPD and KEKEE amino acid sequence motifs, which are present in the GAS R28 protein and the P. falciparum MESA protein, respectively. An LP-KTG cell wall anchor motif (shown in yellow) is located at the carboxy terminus. Numbers in the protein schematic refer to amino acid residues. Reprinted from reference 7 with permission of the publisher.

(Chapter 16) Relationships among GAS phages. Phage sequences present in three GAS genomes were aligned with ClustalW, and an unrooted tree was generated with the Phylip application. Phage sizes (in kilobases) and proven or putative virulence factors encoded by each phage are indicated. Phages that are integrated at the same chromosomal location are color coded in red, green, or blue. Phages that are integrated at unique chromosomal locations are indicated in black. Reprinted from reference 11 with permission of the publisher.

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(Chapter 16) Relationships among GAS phages. Phage sequences present in three GAS genomes were aligned with ClustalW, and an unrooted tree was generated with the Phylip application. Phage sizes (in kilobases) and proven or putative virulence factors encoded by each phage are indicated. Phages that are integrated at the same chromosomal location are color coded in red, green, or blue. Phages that are integrated at unique chromosomal locations are indicated in black. Reprinted from reference 11 with permission of the publisher.

(Chapter 16) Schematics of the prophage and prophage-encoded virulence factor contents of invasive serotype M3 isolates obtained in Ontario, Canada, during 1991–2002. (A) Prophage genotypes are displayed by their prophage and prophage-encoded virulence factor contents per isolate, as follows: top, prophage genotype; bottom, total number of isolates displaying each prophage genotype; and left, specific prophage harbored by the isolate. Prophage-encoded virulence factors per individual prophage (right) are indicated as either present (red) or absent (black). (B) Color-coded stacked columns indicate isolate genotypes as a function of time. Reprinted from reference 12 with permission of the publisher.

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(Chapter 16) Schematics of the prophage and prophage-encoded virulence factor contents of invasive serotype M3 isolates obtained in Ontario, Canada, during 1991–2002. (A) Prophage genotypes are displayed by their prophage and prophage-encoded virulence factor contents per isolate, as follows: top, prophage genotype; bottom, total number of isolates displaying each prophage genotype; and left, specific prophage harbored by the isolate. Prophage-encoded virulence factors per individual prophage (right) are indicated as either present (red) or absent (black). (B) Color-coded stacked columns indicate isolate genotypes as a function of time. Reprinted from reference 12 with permission of the publisher.

(Chapter 17) Structure of modular Cpl-1 lysozyme. (A) Stereo representation of Cpl-1 structure with differently colored domains. Green, catalytic N-terminal domain; orange, linker; cyan, CI domain; magenta, CII domain. Ch molecules are drawn in a ball-and-stick representation. (B) Topology diagram of Cpl-1.Domains are color-coded as in panel A, with the antiparallel β8 strand of the catalytic domain highlighted in orange. In the ChBD, the different Ch binding repeats are labeled. (C) Comparison of amino acid sequences of the Ch binding repeats and the C-terminal tail of Cpl-1.The structural domain, the repeat number, and the corresponding amino acid numbers are shown on the left. Residues in a β-strand conformation are shaded in yellow. Conserved amino acids among the repeats appear in black boxes.The consensus sequence (≥50%) is shown at the bottom, with capital letters indicating 100% conservation. Reprinted from reference 42 with permission of the publisher.

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(Chapter 17) Structure of modular Cpl-1 lysozyme. (A) Stereo representation of Cpl-1 structure with differently colored domains. Green, catalytic N-terminal domain; orange, linker; cyan, CI domain; magenta, CII domain. Ch molecules are drawn in a ball-and-stick representation. (B) Topology diagram of Cpl-1.Domains are color-coded as in panel A, with the antiparallel β8 strand of the catalytic domain highlighted in orange. In the ChBD, the different Ch binding repeats are labeled. (C) Comparison of amino acid sequences of the Ch binding repeats and the C-terminal tail of Cpl-1.The structural domain, the repeat number, and the corresponding amino acid numbers are shown on the left. Residues in a β-strand conformation are shaded in yellow. Conserved amino acids among the repeats appear in black boxes.The consensus sequence (≥50%) is shown at the bottom, with capital letters indicating 100% conservation. Reprinted from reference 42 with permission of the publisher.

(Chapter 19) ds-DNA recombineering intermediate created by the combined action of λ Exo and the single-strand annealing protein, Beta. Exo degrades the 5′ ends of a dsDNA molecule, allowing Beta binding of the 3′ single strands created by the degradation.

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(Chapter 19) ds-DNA recombineering intermediate created by the combined action of λ Exo and the single-strand annealing protein, Beta. Exo degrades the 5′ ends of a dsDNA molecule, allowing Beta binding of the 3′ single strands created by the degradation.

(Chapter 19) Recombineering with dsDNA, illustrating the creation of dsDNA PCR products containing homology for targeting and the use of those products for gene replacement in a recombineering reaction requiring the λ Red functions Exo, Beta, and Gam. (a) Chimeric primers (indicated by green and blue arrows) of ∼70 nt in total length are used for PCR to amplify an antibiotic resistance gene. The 35 to 50 nt at the 5′ ends of the primers are homologous to the regions flanking the gene to be replaced, geneX in this case.The remainder of the primer has homology to amplify the drug cassette. (b) The “ends-out” PCR product is transformed by electroporation into a strain induced for λ Red functions. (c) In vivo recombination replaces geneX with the drug cassette.

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(Chapter 19) Recombineering with dsDNA, illustrating the creation of dsDNA PCR products containing homology for targeting and the use of those products for gene replacement in a recombineering reaction requiring the λ Red functions Exo, Beta, and Gam. (a) Chimeric primers (indicated by green and blue arrows) of ∼70 nt in total length are used for PCR to amplify an antibiotic resistance gene. The 35 to 50 nt at the 5′ ends of the primers are homologous to the regions flanking the gene to be replaced, geneX in this case.The remainder of the primer has homology to amplify the drug cassette. (b) The “ends-out” PCR product is transformed by electroporation into a strain induced for λ Red functions. (c) In vivo recombination replaces geneX with the drug cassette.

(Chapter 19) Use of two-step counterselection to insert a gene fusion, in this case a gfp cassette that will be translationally fused to gene X. In the first recombineering step, a cat sacB cassette is inserted adjacent to gene X, as described in the legend to Color Plate 14, selecting for Cmr. In the second step, a gfp cassette is used to replace the cat sacB cassette, and sucrose-resistant colonies, conferred by the loss of sacB, are selected.These are screened for chloramphenicol sensitivity and the presence of gfp.

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(Chapter 19) Use of two-step counterselection to insert a gene fusion, in this case a gfp cassette that will be translationally fused to gene X. In the first recombineering step, a cat sacB cassette is inserted adjacent to gene X, as described in the legend to Color Plate 14, selecting for Cmr. In the second step, a gfp cassette is used to replace the cat sacB cassette, and sucrose-resistant colonies, conferred by the loss of sacB, are selected.These are screened for chloramphenicol sensitivity and the presence of gfp.

(Chapter 19) Cloning by gap repair into a linear plasmid. A linear plasmid with “ends-in” homology is generated by PCR with chimeric primers as described in the legend to Color Plate 14.The DNA to be inserted into the vector can also be generated by PCR and cotransformed with the linear plasmid (a), or alternatively, a gene or region can be rescued directly from the chromosome onto a plasmid (b). Recombineering restores a circular plasmid that is able to replicate and confers drug resistance, which the linear DNA cannot.

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(Chapter 19) Cloning by gap repair into a linear plasmid. A linear plasmid with “ends-in” homology is generated by PCR with chimeric primers as described in the legend to Color Plate 14.The DNA to be inserted into the vector can also be generated by PCR and cotransformed with the linear plasmid (a), or alternatively, a gene or region can be rescued directly from the chromosome onto a plasmid (b). Recombineering restores a circular plasmid that is able to replicate and confers drug resistance, which the linear DNA cannot.

(Chapter 19) Standard defective λ prophage used by the Court laboratory for recombineering. The Red functions, Exo (α), Beta (β), and Gam (γ), are expressed from the PL operon under the control of the temperature-sensitive CI857 repressor.The strain is propagated at 32°C, except when expression of the Red genes is desired, whereupon Red is induced by shifting the temperature of an exponentially growing culture to 42°C for 15 min.The N antitermination function results in the expression of the Red system.

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(Chapter 19) Standard defective λ prophage used by the Court laboratory for recombineering. The Red functions, Exo (α), Beta (β), and Gam (γ), are expressed from the PL operon under the control of the temperature-sensitive CI857 repressor.The strain is propagated at 32°C, except when expression of the Red genes is desired, whereupon Red is induced by shifting the temperature of an exponentially growing culture to 42°C for 15 min.The N antitermination function results in the expression of the Red system.

(Chapter 19) (a) Minimal defective prophage created by removal of the N through kil genes in the PL operon and replacement of rexA and rexB with a drug resistance cassette, either ampicillin (bla) or chloramphenicol (cat). With this system, raising the temperature induces the operon directly without N-mediated antitermination. (b) Minimal prophage moved onto a high-copy-number vector. The pBR322 origin of DNA replication between nucleotide coordinates 2348 and 3296 was amplified.This linear PCR product was used to clone the minimal prophage in a gap repair reaction. This linear pBR322 fragment contains ori but lacks an antibiotic resistance gene, so only those plasmid clones that have undergone successful recombineering will contain an antibiotic resistance marker inherited from the prophage.The high-copy-number plasmids thus generated, pSIM2 and pSIM4 (Cmr and Ampr, respectively),were used as targets in subsequent recombineering reactions.The pBR322 segment was replaced precisely with a linear DNA containing a temperature-sensitive pSC101 origin and the rep gene.The recombinant conferring drug resistance was selected on a polA host, in which the pBR322 origin does not replicate. Other plasmid origins (Table 2) were used in a similar manner to replace the pBR322 origin segment.

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(Chapter 19) (a) Minimal defective prophage created by removal of the N through kil genes in the PL operon and replacement of rexA and rexB with a drug resistance cassette, either ampicillin (bla) or chloramphenicol (cat). With this system, raising the temperature induces the operon directly without N-mediated antitermination. (b) Minimal prophage moved onto a high-copy-number vector. The pBR322 origin of DNA replication between nucleotide coordinates 2348 and 3296 was amplified.This linear PCR product was used to clone the minimal prophage in a gap repair reaction. This linear pBR322 fragment contains ori but lacks an antibiotic resistance gene, so only those plasmid clones that have undergone successful recombineering will contain an antibiotic resistance marker inherited from the prophage.The high-copy-number plasmids thus generated, pSIM2 and pSIM4 (Cmr and Ampr, respectively),were used as targets in subsequent recombineering reactions.The pBR322 segment was replaced precisely with a linear DNA containing a temperature-sensitive pSC101 origin and the rep gene.The recombinant conferring drug resistance was selected on a polA host, in which the pBR322 origin does not replicate. Other plasmid origins (Table 2) were used in a similar manner to replace the pBR322 origin segment.

(Chapter 20) Comparison of the genomes of phages SP6 and K1-5.The blue scale represents percentages of amino acid identity. Both phages are very similar at both the sequence level and the genome organization level, with the exception of the tail genes that confer specificity for different capsules.

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(Chapter 20) Comparison of the genomes of phages SP6 and K1-5.The blue scale represents percentages of amino acid identity. Both phages are very similar at both the sequence level and the genome organization level, with the exception of the tail genes that confer specificity for different capsules.